Regulation of antibody production by B-1 cells Summary Natural IgM provides a significant first-line defense against pathogen invasion. It also contributes to immune and tissue homeostasis by preventing the development of autoreactive B cells and by contributing to the removal of cell debris. Despite its important functions, what regulates natural antibody production is insufficiently understood. We recently showed that natural IgM secretion occurs predominantly in spleen and bone marrow, and is contributed by at least two distinct populations of B-1 cells: B-1-derived (CD19- IgM+ CD43+ Blimp-1+) plasma cells (B-1PC) and ?classical? (CD19+ IgM+ CD43+ Blimp-1neg) B-1 cells. Surprisingly, the latter population neither expressed, nor appeared to require Blimp-1 for IgM production, the master regulator of plasma cell differentiation. In other words, IgM-secreting B-1 cells did not seem to be differentiation-intermediates between non-secreting Blimp-1neg B-1 and Blimp-1hi B-1PC. The data explain why B cell-specific Blimp-1-deficient mice continue to generate significant amounts of IgM, but not most other Ig, except IgG3, another Ig-subtype mainly generated by B-1 cells. The underlying mechanisms and transcriptional pathways that regulate Blimp-1- independent antibody secretion, however, have not been revealed. The overall objective of our work is to elucidate the regulation of natural antibody production, in order to harness their immune-protective functions for prophylactic and/or therapeutic uses in the future. The objective of this application is to determine the differentiation pathways of all natural IgM-secreting B-1 cells. We hypothesize that differentiation to either natural IgM secreting Blimp-1neg B-1 cells or Blimp-1+ B-1PC occur via two distinct differentiation pathways induced by activating B-1 cell subsets that emerge at different times in ontogeny. We propose two Specific Aims to test this hypothesis. In Aim 1 we will determine the cellular origins of B-1 and B-1PC by reconstituting neonatal B cell- deficient chimeras with B-1 cell precursors taken at distinct times in ontogeny. B-1 lineage tracing studies will probe further for heterogeneity among B-1 cells, and repertoire analysis will link developmental paths to specificity. Aim 2 is to assess the transcriptional profile of all IgM-secreting B-1 and B-1PC at the single-cell level by conducting 10x Genomics single-cell expression profiling of FACS-purified bone marrow and spleen cells. Genes known to be differentially expressed by IgM-secreting and non-secreting B-1 cells and B-1PC will aid identification of their cell clusters and identify any potential additional B-1 clusters. Data mining will identify candidate Blimp-1 independent differentiation pathways, which we would explore in detail in follow-up work. Expected results would identify novel candidate transcriptional pathways regulating B-1 cell differentiation and natural Ig secretion for in-depth future functional exploration. Given the similarities in Blimp-1-dependent T and B cell effector differentiation, a new Blimp-1 independent differentiation path may similarly be shared broadly among other cells, and thus be of high significance beyond the regulation of natural Ig production.